The present invention relates to a device and method for detecting the concentration of biological materials, in particular a TSH (Thyroid Stimulating Hormone), in a sample.
Typical procedures for analyzing biological materials, such as nucleic acid, protein, lipid, carbohydrate, and other biological molecules, involve a variety of operations starting from raw material. These operations may include various degrees of cell separation or purification, cell lysis, amplification or purification, and analysis of the resulting amplification or purification products.
As an example, in DNA-based blood analyses, samples are often purified by filtration, centrifugation or electrophoresis so as to eliminate all the non-nucleated cells, which are generally not useful for DNA analysis. Then, the remaining white blood cells are broken up or lysed using chemical, thermal or biochemical means in order to liberate the DNA to be analyzed. Next, the DNA is denatured by thermal, biochemical or chemical processes and amplified by an amplification reaction, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), and the like. The amplification step allows the operator to avoid purification of the DNA being studied because the amplified product greatly exceeds the starting DNA in the sample.
If RNA is to be analyzed the procedures are similar, but more emphasis is placed on purification or other means to protect the labile RNA molecule. RNA is usually copied into DNA (cDNA) and then the analysis proceeds as described for DNA.
Finally, the amplification product undergoes some type of analysis, usually based on sequence or size or some combination thereof. In an analysis by microarray hybridization, for example, the amplified DNA is passed over a plurality of detectors made up of individual oligonucleotide detector fragments that are anchored, for example, on electrodes. If the amplified DNA strands are complementary to the oligonucleotide detectors or probes, stable bonds will be formed between them (hybridization) under specific temperature conditions. The hybridized detectors can be read by observation using a wide variety of means, including optical, electromagnetic, electromechanical or thermal means.
Other biological molecules are analyzed in a similar way, but typically molecule purification is substituted for amplification, and detection methods vary according to the molecule being detected. For example, a common diagnostic involves the detection of a specific protein by binding to its antibody. Such analysis requires various degrees of cell separation, lysis, purification and product analysis by antibody binding, which itself can be detected in a number of ways. Lipids, carbohydrates, drugs and small molecules from biological fluids are processed in similar ways.
Today, immunoassays of all kinds dominate the in vitro diagnostic (IVD) market. In 2005, according to Kalorama Information (New York City), the global immunoassay market generated 5.8 billion US$ in total revenues. New growth in immunoassays is tied to the development of autoimmune, cardiac, and tumor markers that play a significant role in disease diagnosis and monitoring. By 2010, the immunoassay market is expected to reach 8.1 billion US$ with an annual growth rate of 7%.
In the following, reference will be made to the measure of proteins/carbohydrates in a biological liquid, typically serum or urine, in biochemical tests called immunoassays that use the reaction of the proteins/carbohydrates to one or more antibodies as a basis for the assay measurement.
More particularly, in the following reference will be made to the measure of the concentration of the Thyroid Stimulating Hormone (TSH), but similar considerations and same methods and apparatuses apply to a variety of biological assays, e.g., for the measure of glycosylated hemoglobin and carbohydrate deficient transferrin (CDT) (the latter test being usable to discriminate between occasional and chronic drinking).
As known, the thyroid gland produces hormones that control the rate of metabolism and affect the development and operability of many other body functions. The two most common thyroid disorders are hyperthyroidism (overactive thyroid) and hypothyroidism (under active thyroid). Thyroid stimulating hormone (TSH) produced by the anterior pituitary gland regulates the production of two hormones (T3—triiodothyronine and T4—thyroxine) from the thyroid in a negative feedback mechanism. When levels of T3 and T4 are low, TSH is stimulated to produce more and more T3 and T4. Similarly, when levels are high, TSH production is decreased, which in turn decreases T4 and T3 levels.
In the past, the measure of the concentration of TSH was made using a first generation radio-immunological assay that had quite low sensitivity and was not able to discriminate low values, still within in the normal range, from slightly lower ones, correlating with hyperthyroidism.
Around the mid-eighties, second generation immunologic techniques were developed that used two anti-TSH antibodies, and the dual antibody system had somewhat improved the sensitivity. Later, in the nineties, these techniques were again improved to arrive at third generation methods that have a much higher sensitivity and allow the measure of TSH also for patients having serologic atypias tied to different thyroid diseases. Third generation TSH tests are solid phase enzyme-linked immunosorbent assays, using a mouse monoclonal anti-TSH antibody for solid phase immobilization and goat anti-TSH antibody linked to usually horseradish peroxidase, thus allowing signal amplification.
Third generation methods have a functional sensitivity (meaning therewith the lowest concentration allowing the dosage to maintain a dosage variation coefficient of about 20% or less) of about 0.01 to 0.02 μl U/mL and thus are able to provide quite precise results for hyperthyroid patients.
Traditional immunoassays, such as, e.g., ELISA (Enzyme-Linked ImmunoSorbent Assay) are based on the use of primary antibodies, together with enzymatic second antibodies and associated substrates to generate a final signal that may be compared with known thresholds.
These known solutions are fully manual, in that they require manual insertion of the samples, antibodies, and washing liquids and require an optical reader; in addition they require skilled personnel to perform the various binding and washing steps, so that they only allow clinical testing in central laboratories. In addition, they are complex, involve time-consuming procedures, and use potentially hazard and expensive materials.